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Arquivos de Gastroenterologia

Print version ISSN 0004-2803

Arq. Gastroenterol. vol.51 no.2 São Paulo Apr./June 2014

https://doi.org/10.1590/S0004-28032014000200012 

Original Articles

LOW DOSE CAPSULE BASED 13C-UREA BREATH TEST COMPARED WITH THE CONVENTIONAL 13C-UREA BREATH TEST AND INVASIVE TESTS

Teste respiratório com 13C-uréia em cápsula de baixa dose em comparação com o teste respiratório com 13C-uréia convencional e testes invasivos

Rejane MATTAR1 

Cibele Aparecida VILLARES1 

Priscila Faria França MAROSTEGAM2 

Cleuber Esteves CHAVES2 

Vanusa Barbosa PINTO2 

Flair José CARRILHO1 

1Divisão de Gastroenterologia e Hepatologia. Hospital das Clinicas da Faculdade de Medicina de São Paulo – FMUSP

2Divisão de Farmácia. Hospital das Clinicas da Faculdade de Medicina de São Paulo – FMUSP, São Paulo, SP, Brasil


ABSTRACT

Context

One of the limitations of 13C-urea breath test forHelicobacter pylori infection diagnosis in Brazil is the substrate acquisition in capsule presentation.

Objectives

The purpose of this study was to evaluate a capsule-based13C-urea, manipulated by the Pharmacy Division, for the clinical practice.

Methods

Fifty patients underwent the conventional and the capsule breath test. Samples were collected at the baseline and after 10, 20 and 30 minutes of 13C-urea ingestion. Urease and histology were used as gold standard in 83 patients.

Results

In a total of 50 patients, 17 were positive with the conventional13C-urea (75 mg) breath test at 10, 20 and 30 minutes. When these patients repeated breath test with capsule (50 mg), 17 were positive at 20 minutes and 15 at 10 and 30 minutes. The relative sensitivity of13C-urea with capsule was 100% at 20 minutes and 88.24% at 10 and at 30 minutes. The relative specificity was 100% at all time intervals. Among 83 patients that underwent capsule breath test and endoscopy the capsule breath test presented 100% of sensitivity and specificity.

Conclusions

Capsule based breath test with 50 mg 13C-urea at twenty minutes was found highly sensitive and specific for the clinical setting. HEADINGS- Helicobacter pylori. Breath Test. Urea, analysis.

Key words: Helicobacter pylori ; Breath Test; Urea, analysis

RESUMO

Contexto

Uma das limitações para o teste respiratório com13C-uréia ser incorporado na prática clínica no Brasil para diagnóstico de infecção pelo Helicobacter pylori(H. pylori) é a aquisição do substrato em apresentação de cápsula.

Objetivos

O objetivo deste estudo foi avaliar a utilidade de13C-uréia em cápsula, manipulada pela Divisão de Farmácia de um Hospital terciário para a prática clínica.

Métodos

Cinquenta pacientes foram submetidos ao teste respiratório convencional com 75mg de 13C-uréia e ao teste com cápsula de 50 mg de 13C-uréia. Amostras de ar expirado foram coletadas no basal e após 10, 20 e 30 minutos da ingestão de 13C-uréia para definição do melhor tempo de coleta. A urease e a histologia foram usadas como padrão ouro em 83 pacientes que se submeteram ao teste respiratório com único ponto de coleta.

Resultados

Num total de 50 pacientes, 17 foram positivos com teste respiratório convencional com 75 mg de 13C-uréia aos 10, 20 e 30 minutos. Quando esses pacientes repetiram o teste respiratório com13C-uréia em cápsulas de 50 mg, 17 foram positivos aos 20 minutos (Índice Kappa 1,0; P<0,05) e 15 aos 10 e 30 minutos (Índice Kappa 0,90; P<0,05). A sensibilidade relativa da 13C-uréia em cápsulas de 50 mg foi 100% aos 20 minutos e 88,24% aos 10 e 30 minutos. A especificidade relativa foi de 100% em todos os intervalos de tempo. Entre 83 pacientes que se submeteram ao teste respiratório com cápsula e endoscopia, aqueles que eram H. pylori positivos tiveram teste respiratório positivo e aquelesH. pylori negativo tiveram teste respiratório negativo, mostrando sensibilidade e especificidade de 100%.

Conclusões

O teste respiratório com cápsula de 50 mg de 13C-uréia aos 20 minutos foi considerado altamente sensível e específico para a prática clínica.

Palavras-Chave: Helicobacter pylori ; Testes respiratórios; Ureia, análise

Introduction

Helicobacter pylori (H. pylori) infection is the main etiological cause of chronic gastritis, peptic ulcer disease(19) and its recurrence(17). The eradication of H. pyloriinfection has been considered primary prophylaxis of gastric cancer, and responsible for gastric cancer reduced incidence and mortality(14); thus, diagnosis of H. pylori infection is highly important for the correct management of these disorders(17).

Among the noninvasive tests for H. pylori infection diagnosis, urea breath test using urea labeled with carbon-13, non-radioactive isotope, was originally described by Graham et al. in 1987(10), showing excellent correlation with culture and histological analysis. Marshall and Surveyor in the next year(20) reported also a simple breath test with urea labeled with carbon-14, presenting a significant difference between individuals that were H. pylori positive in relation to the controls. The principle of urea labeled breath test is the production of urease by H. pyloriin the acidic environment of the stomach that hydrolyses urea in carbon labeled bicarbonate that is absorbed and, released as carbon dioxide (13CO2 or 14CO2) in the exhaled breath(24). We had previously validated14C-urea breath test with 100% sensitivity and specificity(21); nonetheless, due to its restrictions to pregnant woman and children, the conventional 13C-urea breath test(30)was further introduced for the clinical practice.

The accuracy of 13C-urea breath test is very high with sensitivity of 88% to 95% and specificity of 95 to 100%, being an acceptable non-invasive test to diagnose H. pylori infection for initial diagnosis and eradication control after treatment(17). In Brazil, the conventional test with 13C-urea in powder dissolved in orange juice has been widely performed in children with doses of 50 mg and 75 mg in patients with less than 30 kg and more than 30 kg, respectively(3, 15, 16, 29), and in adults(30). However, the conventional test with liquid13C-urea in orange juice may suffer the influence of urease-producing oral flora, increasing 13CO2 excretion in earlier sample collections with false-positive results, also a higher dose is necessary(7, 25, 27).

Capsule based 13C-urea breath test has been reported with lower dose than the conventional test with high sensitivity and specificity, avoiding bacterial oral flora interference, and in a shorter time(7, 25, 27, 28). One of the limitations for 13C-urea breath test to be incorporated into daily gastroenterology practice in Brazil is the substrate (13C-urea) acquisition in capsule presentation(6). Thus, the purpose of this study was to evaluate the usefulness of a capsule-based 13C-urea, manipulated by the Pharmacy Division of a Tertiary Care Hospital for the clinical practice.

Methods

Subjects

This study was approved by the local Ethics Committee. The inclusion criteria were patients who had not taken antibacterial therapy for at least one month and proton pump inhibitors for less than 15 days prior the urea breath test. Patients that underwent H. pylori infection eradication regimen for more than one month and those of initial H. pyloriinfection diagnosis were included in the study. In the first phase of the study fifty patients (mean age of 42.52±11.81 years; thirty-five were women) who underwent 13C-urea breath test were invited to participate, and gave written informed consent. The purpose of the first phase was to establish the best point time collection of capsule based breath test.

In the second phase of the study after the time point collection was set, 13C-urea breath test was validated using urease and histology for H. pylori status determination. Cases were considered positive when urease and histology were positive. For this analysis 83 patients (mean age of 56.13±12.89 years; 45 were women) that underwent upper gastrointestinal endoscopy and capsule based13C-urea breath test entered the study. Cases that had discordant results between histology and urease tests were excluded. One patient with bleeding gastric ulcer that wasH. pylori negative by urease and histology and H. pylori positive by urea breath test (21.3 dob) was excluded.

Conventional 13C-urea breath

The procedure was performed according to technique previously described(30). Briefly, patients had to fast at least 4-hour before the test and not to smoke or drink sparkling water on the day of the test. Breath sample was collected in aluminized bag as the baseline value, followed by ingestion of 75 mg of 13C-urea (Euriso-top®, France) dissolved in 200 mL of orange juice (pH = 3.0; powdered orange juice in packets prepared with water immediately before use, Dia Brasil Sociedade Ltda., Brazil). Breath samples were collected after 10, 20 and 30 minutes of 13C-urea ingestion, and analyzed by infrared spectroscopy (IRIS Doc, Wagner Analysen, Germany). A DOB (Delta over baseline-value) ≥4.0‰ was considered positive for H. pyloriinfection according to the manufacturer instruction and previous reports in Brazil(3, 15, 16, 29, 30).

Capsule based urea breath test

Capsules were prepared by the Pharmacy Division of Hospital das Clínicas da FMUSP. The formulation consisted of 50 mg of 13C-urea (Euriso-top®, France) and 170 mg of citric acid that were mixed by trituration, reducing all to a fine and uniform powder and filled in hard gelatin capsule size 4, using capsule hand-filling machine (Capsutec, Brazil)(9). All batches of 50 mg of13C-urea capsules were tested for weight variation. Each capsule weight was between 90 per cent and 110 per cent of the average and with the relative standard deviation less than or equal to 5.0%(1). The capsules were packaged into pouch unit-dose and kept in cardboard boxes with capsules of silica gel(32). The expiration date of the capsules prepared was 30 days. Not exceeding the period of one week, the patients repeated breath test, ingesting capsules of 50 mg of 13C-urea with 200 mL of orange juice. Breath samples were collected at basal, and after 10, 20 and 30 minutes of capsule ingestion and analyzed by infrared spectroscopy (IRIS Doc, Wagner Analysen, Germany). A DOB (Delta over baseline-value) ≥4.0‰ was considered positive for H. pylori infection.

Urease test

The biopsy samples taken from the antrum and corpus of the stomach were inserted into the homemade urease test tubes according to the previously described technique. The urease test tube was examined over the next 24 hours(22).

Histology

Gastric biopsy samples from the antrum and corpus were fixed in 10% formalin and stained with hematoxylin and eosin (H&E) and Giemsa forH. pylori identification, as was previously described(23).

Statistical Analyses

The sample size of 42 patients was calculated through a test between a null hypothesis given by Kappa statistic equal to 0.4 (fair) and an alternative hypothesis with Kappa statistic equal to 0.8 (substantial) for a prevalence of 53% of H. pylori in the studied population(18), considering a statistical significance of 5% and a power of 80%.

Kappa Index measure of agreement(31) was performed with SPSS software version 15.0 for Windows (Chicago, Illinois, USA). The diagnostic performance of capsule based breath test was estimated with the software R version 2.15.2 (R Core Team, R Foundation for Statistical Computing Vienna, Austria, http://www.R-project.org). Relative sensitivity and relative specificity were determined based on subjects with positive conventional urea breath test and subjects with negative conventional urea breath test, as the true-positives and the true-negatives, respectively(11). Breath 13CO2 values were compared between patients H. pylori positive with those H. pylori negative by Mann-Whitney on non-normal distributions, or by Student’s t-test on normal distribution. The analysis of capsule based breath test in relation to the H. pylori status (urease and histology) was performed by Fisher’s exact test. Sensitivity, specificity, positive predictive value and negative predictive value were estimated, using urease and histology as gold standard. A value ofP<0.05 was considered significant.

Results

In a total of 50 patients, 17 were positive with the conventional 13C-urea (75 mg) breath test at 10, 20 and 30 minutes. When these patients repeated the 13C-urea breath test with 50 mg capsules, 17 were positive at 20 minutes (Kappa Index 1.0 = almost perfect;P<0.05) and 15 at 10 and 30 minutes (Kappa Index 0.90 = almost perfect; P<0.05) (Figure 1).

FIGURE 1. Comparison of the breath test results obtained with 50 mg13C-urea capsules (C) and with the conventional 75 mg13C-urea (P) at 10, 20 and 30 minute (m) time point collection 

The relative sensitivity of 13C-urea with 50 mg capsules was 100% (95%CI: 77.92%-100%) at 20 minutes and 88.24% (95%CI: 64.16%-97.75%) at 10 and at 30 minutes. The relative specificity was 100% (95%CI: 87.31%-100%) at all time intervals.

Breath 13CO2 values were significantly higher (P<0.05) in those patients H. pyloripositive than in those H. pylori negative. There was a striking difference of the mean 13CO2 excretion between H. pylori positive and H. pylori negative groups (Table 1), only at 10 (DOB = 2.20‰ and DOB = 2.80‰) and 30 (DOB = 2‰ and DOB = 3.10‰) minutes, four H. pyloripositive patients presented DOB values lower than the cutoff of 4‰ with capsule based breath test (Figure 1). For 50 mg 13C-urea using capsule, cutoff value of DOB≥2‰ gave 100% relative sensitivity and specificity at 10, 20 and 30 minutes. However, at 20 minutes these patients had positive breath test; hence, this time point collection was considered the most reliable for 50 mg capsule based breath test.

TABLE 1. Mean, standard deviation, minimum and maximum values of conventional and capsule based breath test at 10, 20 and 30 minutes of the positive and negative H. pylori subjects, and of the 83 patients that underwent breath test with 50 mg capsule at 20 minutes for breath test validation against gold standard 

Test minutes Mean SD Minimum Maximum Mean SD Minimum Maximum
Hp+ Hp+ Hp+ Hp+ Hp- Hp- Hp- Hp-
Cap 10 13.80 12.67 2.20 50.9 -0.69 1.16 -4 1.20
P 10 26.61 20.64 6.50 81.20 0.08 1.12 -3 2.20
Cap 20 21.42 16.32 4.80 62.80 -0.58 1.38 -4 1.90
P 20 27.25 16.8 9.40 64 0.29 1.44 -5 3.00
Cap 30 17.48 12.61 2 44.20 -0.66 1.61 -5 1.90
P 30 21.49 13.06 5.50 58.90 -0.27 1.22 -3 2.50
83 pat 21.57 12.84 4.5 51.9 0.33 0.6 0.1 3.6

Helicobacter pylori; SD: standard deviation; Cap: capsule based breath test; P: conventional breath test; pat, patients.

When capsule based breath test was validated against a gold standard (urease and histology), 32 patients that were H. pylori positive had positive urea breath test and 51 patients that were H. pylorinegative had negative breath test; thus, the sensitivity (95%CI: 86.96%-100%) and specificity (95%CI: 91.42%-100%) were 100%. The demographic and endoscopic findings of these 83 patients are presented in Table 2, 52 (62.7%) patients had peptic ulcer, 28 (33.7%) had gastritis, 2 (2.4%) had atrophic gastritis and 1 (1.2%) had normal endoscopy. The statistical analysis of the endoscopic findings in relation to the H. pyloristatus, gender and ethnicity was not significant. The mean13CO2 excretion of H. pylori positive patients was DOB = 21.57±12.84‰, and of H. pylori negative patients was DOB = 0.33±0.6‰ (Table 1), the difference between these two groups was significant (P<0.05).

TABLE 2. Demographic and endoscopic findings of the 83 patients that performed13C-urea breath test with 50 mg capsule at 20 min time point collection (%) 

Endoscopy Normal Gastritis Peptic ulcer Atrophic gastritis Total
H. pylori neg. 1 (2) 13 (25.5) 35 (68.6) 2 (3.9) 51 (61.4)
H. pylori pos.   15 (46.9) 17 (53.1)   32 (38.6)
White 1 (1.4) 25 (36.2) 41 (59.4) 2 (2.9) 69 (83.1)
Brown   1 (12.5) 7 (87.5)   8 (9.6)
Black   1 (25) 3 (75)   4 (4.8)
Japanese-Brazilian   1 (50) 1 (50)   2 (2.4)
Men   13 (34.2) 25 (65.8)   38 (45.8)
Women 1 (2.2) 15 (33.3) 27 (60) 2 (4.4) 45 (54.2)

Four patients that were H. pylori positive and were taking omeprazole until the day of the urea breath test gave positive results as follows: DOB = 7.6‰, 8.1‰, 24.1‰ and 37.7‰.

Discussion

According to the Maastricht IV Consensus report(17) urea breath test and stool antigen testing are acceptable non-invasive tests for H. pylori infection diagnosis with 88%-95% sensitivity and 95%-100% specificity. In Brazil, commercial capsule of 13C-urea is not available, hindering the incorporation of this test to the daily gastroenterology practice(6). The Pharmacy Division of our Institution may formulate capsule of 13C-urea, and provide for the patients of our Hospital to undergo urea breath test.

The comparison of capsule based breath test with the conventional test showed that citric acid used as excipient of the capsule may help acidifying the stomach milieu, inducing H. pylori for the urease synthesis(13). There is no interference of the oral flora, as the capsule is swallowed and disintegrated directly in the stomach. Capsules prevented the urea to be released before reaching the stomach, requiring a lower dose of 13C-urea(7, 13, 25, 27).

In this report, we showed that 13C-urea breath test performed with formulated 50 mg hard gelatin capsule with citric acid and taken with orange juice (pH = 3.0) had almost perfect agreement (Kappa Index measure of agreement = 1;P<0.05) with the conventional 13C-urea breath test in powder dissolved in orange juice. The conventional breath test with 75 mg of13C-urea in 200 mL orange juice and considering a cutoff value of DOB≥4‰ was previously validated with 100% sensitivity and specificity(4), and performed in other publications in Brazil(3, 15, 16, 29, 30).

Considering a cutoff value of DOB≥4‰ at 20 minute time collection or a cutoff value of DOB≥2‰ at 10, 20 and 30 minutes, the capsule (50 mg) urea breath test had 100% relative sensitivity and relative specificity. A cutoff value of DOB≥4‰ at 20 minute time collection was chosen for the clinical setting, as was previously demonstrated that a change in the cutoff value within 2 and 5 would have little effect on the clinical accuracy of the test, as positive and negative results tend to group outside this range(12).

In this study powdered orange juice (pH = 3.0) was taken with the capsule to increase sensitivity of the test, although citric acid as excipient of the capsule formulation may provide an acidic microenvironment. The activity of bacterial urease and the 13CO2 excretion were significantly higher with apple juice (pH = 3.0) or with acidified meal (pH = 3.0) than with neutral meal, and independent of gastric emptying(26). Other authors(7, 13, 27) that tested 50 and or 100 mg capsule of 13C-urea with water, showed sensitivity of 95%-100% and specificity of 97.6%-100%, suggesting that acid test meal may be omitted, lowering the cost of the exam. Nonetheless, powdered orange juice had low cost, long expiration date, and stability. Citric acid solution instead of juice showed high sensitivity (89.1%-100%) and specificity (100%) with low concentrations of 10 to 25 mg 13C-urea solution(8). Conversely, exam performed with 25 mg13C-urea solution in orange juice had lower sensitivity and specificity, and was not recommended for the clinical setting in our country(5).

Previously was shown that even after 7-day treatment with omeprazole, no patients had false negative results with the tablet based breath test (two tablets of 50 mg 13C-urea and 463 mg citric acid)(13). In this report four patients that did not stop taking omeprazole also had positive breath test. This is an important finding, as some patients with continuous proton pump inhibitor therapy may not discontinue medication with risk of gastrointestinal bleeding(2). Although the recommendation is to avoid all proton pump inhibitors and H2-receptor antagonists, among omeprazole, lansoprazole and pantoprazole, the latter did not interfere in the accuracy of13C-urea breath test. In addition, H2-receptor antagonists, that increase intragastric pH, have no inhibitory effect on H. pylori density, different from proton pump inhibitors that decreases the density of H. pylori on the stomach surface(12). Thus, the false negative effect of H2-receptor antagonists and proton pump inhibitors on capsule 13C-urea breath test and the real necessity of discontinuing these medications should be further evaluated. Although the effects of increasing gastric pH by H2-receptor antagonists and sodium bicarbonate solution and of density of H. pylori were already explored during13C-urea breath test in solution by Graham et al.(12) that concluded H2-receptor antagonists differ from proton pump inhibitors as high intragastric pH may cause a reduction in urease activity, unrelated to a reduced bacterial load and reversed by citric acid.

When capsule based breath test was validated against a gold standard (urease and histology), the sensitivity and the specificity were high and comparable to previous reports with 13C-urea in capsule presentation that showed sensitivity of 95%-100% and specificity of 92-100%(7, 13, 25, 27, 28).

In conclusion, capsule based breath test with 50 mg 13C-urea at 20 minutes was found suitable for the clinical setting with 100% sensitivity and specificity.

ACKNOWLEDGMENT

The authors thank Marcio Augusto Diniz from the Laboratory of Epidemiology and Statistics of the Department of Gastroenterology, School of Medicine - University of São Paulo for the statistical analysis, Joyce Matie Kinoshita da Silva-Etto for technical support, Maria de Fátima Silva Miyamoto for the manipulation, and the Division of Pathology, Gastrointestinal Endoscopy and Center of diagnosis in Gastroenterology - CDG of Hospital das Clínicas da FMUSP for the histological analysis and endoscopies procedures, respectively. This study was supported by Fundação Faculdade de Medicina and Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo.

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Received: January 16, 2014; Accepted: February 26, 2014

Correspondence: Rejane Mattar. Av. Dr. Enéas de Carvalho Aguiar 255, 9° andar sala 9159, CEP: 05403-000 - São Paulo, SP, Brasil. E-mail:rejane-mattar@ig.com.br

Declared conflict of interest of all authors: none

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